The present invention relates to Tek antagonists and to the use of Tek antagonists to inhibit angiogenesis or other Tek-mediated responses in a mammal.
A. Angiogenesis
Angiogenesis, the generation of new blood vessels, is a spatially and temporally regulated process in which endothelial cells proliferate, migrate, and assemble into tubes, in response to endogenous positive and negative regulatory molecules. Angiogenesis plays important roles in both normal and pathological physiology.
Under normal physiological conditions, angiogenesis is involved in fetal and embryonic development, wound healing, organ regeneration, and female reproductive remodeling processes including formation of the endometrium, corpus luteum, and placenta. Angiogenesis is stringently regulated under normal conditions, especially in adult animals, and perturbation of the regulatory controls can lead to pathological angiogenesis.
Pathological angiogenesis has been implicated in the manifestation and/or progression of inflammatory diseases, certain eye disorders, and cancer. In particular, several lines of evidence support the concept that angiogenesis is essential for the growth and persistence of solid tumors and their metastases (see, e.g., Folkman, N. Engl. J. Med. 285:1182, 1971; Folkman et al., Nature 339:58, 1989; Kim et al., Nature 362:841, 1993; Hori et al., Cancer Res., 51:6180, 1991). Angiogenesis inhibitors are therefore being tested for the prevention (e.g., treatment of premalignant conditions), intervention (e.g., treatment of small tumors), and regression (e.g., treatment of large tumors) of cancers (see, e.g., Bergers et al., Science 284:808, 1999).
Although several anti-angiogenic agents are presently under development and testing as therapeutics, there is a need for additional methods of inhibiting angiogenesis for the prevention, abrogation, and mitigation of disease processes that are dependent on pathological angiogenesis.
B. Tek Polypeptides
The receptor tyrosine kinases (RTKs) are a large and evolutionarily conserved family of proteins involved in the transduction of extracellular signals to the cytoplasm. Among the RTKs believed to be involved in vascular morphogenesis and maintenance are the vascular endothelial growth factor (VEGF) receptors and Tek (see Hanahan, Science 277:48, 1997).
Tek, which has also been called Tie2 and ork, is an RTK that is predominantly expressed in vascular endothelium. The molecular cloning of human Tek (ork) has been described by Ziegler, U.S. Pat. No. 5,447,860. Four Tek ligands, angiopoietin-1, angiopoietin-2, angiopoietin-3, and angiopoietin-4 (Ang1, Ang2, Ang3, and Ang4), have been described (Davis et al., Cell 87:1161, 1996; Maisonpierre et al., Science 277:55, 1997; Valenzuela et al., Proc. Natl. Acad. Sci. USA 96:1904, 1999). These ligands have distinct expression patterns and activities with respect to Tek. xe2x80x9cTie ligand homologuesxe2x80x9d designated NL1, NL5, NL8, and NL4 are described in U.S. Pat. No. 6,057,435.
Tek knockout mice have defects in vascular development, and die during embryogenesis (see Dumont, Genes Dev. 8:1897, 1994; Sato, Nature 376:70, 1995), suggesting that Tek plays a role in the development of embryonic vasculature.
Lin et al. have described a soluble Tek (Tie2) inhibitor designated ExTek.6His, consisting of the entire extracellular portion of murine Tek fused to a six-histidine tag (J. Clin. Invest. 100(8):2072, 1997; WO 98/18914). ExTek.6His inhibited growth and tumor vascularization in a rat cutaneous window chamber model, and blocked angiogenesis stimulated by tumor cell conditioned media in a rat corneal micropocket assay. Peters et al. have also described a replication-defective adenoviral vector designated AdExTek, which expresses the murine Tek extracellular domain (Proc. Natl. Acad. Sci. USA 95:8829, 1998; WO 98/18914). AdExTek inhibited the growth and metastasis of a murine mammary carcinoma and a murine melanoma.
While ExTek.6His and AdExTek may prove useful as anti-angiogenic agents, there is a need for additional and improved Tek antagonists and additional and improved methods of inhibiting angiogenesis or other Tek-mediated responses using Tek antagonists.
The present invention provides Tek antagonists and methods of using Tek antagonists to inhibit angiogenesis or other Tek-mediated responses in a mammal in need of such treatment. The invention is based in part on the unexpected discovery that fragments of the Tek extracellular domain, lacking all or part of the region containing fibronectin type III (FNIII) motifs, can have a higher binding affinity for Tek ligands than polypeptides comprising full length Tek extracellular domain.
In some preferred embodiments the Tek antagonist is a polypeptide comprising a fragment of Tek extracellular domain, wherein the fragment lacks all or part of the region containing fibronectin type III (FNIII) motifs and wherein the polypeptide retains the ability to bind at least one Tek ligand. In preferred embodiments the fragment lacks at least residues 473-745 of the Tek extracellular domain; in more preferred embodiments the Tek ligand is angiopoietin-1, angiopoietin-2, or angiopoietin-4. In most preferred embodiments, the Tek antagonist is a polypeptide that has a higher binding affinity for a Tek ligand than does a polypeptide comprising full length Tek extracellular domain.
The invention also encompasses nucleic acids encoding polypeptides according to the invention, and polypeptides produced by expressing such a nucleic acid in a recombinant host cell under conditions that permit expression of the polypeptide.
In some preferred embodiments, the Tek antagonist is a soluble Tek multimer, preferably a dimer or trimer, and most preferably comprising an Fc polypeptide or a leucine zipper. The Tek is preferably human Tek. In some preferred embodiments the soluble Tek multimer comprises a fragment of Tek extracellular domain, wherein the fragment lacks all or part of the region containing fibronectin type III (FNIII) motifs and wherein the polypeptide retains the ability to bind at least one Tek ligand. In some preferred embodiments the soluble Tek multimer comprises residues 23-472 or 23-704 of SEQ ID NO:2.
The invention also encompasses antibodies or antibody fragments that bind specifically to a polypeptide according to the invention, and antibodies or antibody fragments that are capable of competitively inhibiting the binding of a Tek ligand to a polypeptide according to the invention. The antibodies are preferably selected from the group consisting of monoclonal antibodies, humanized antibodies, transgenic antibodies, and human antibodies.
The invention also provides methods of inhibiting angiogenesis or other Tek-mediated responses in a mammal in need of such treatment, comprising administering to the mammal an inhibition-effective amount of a Tek antagonist. The Tek antagonist is preferably a fragment of Tek extracellular domain, a soluble Tek multimer, or an antibody or antibody fragment. In some preferred embodiments the Tek antagonist is administered in a composition comprising a pharmaceutically acceptable carrier.
The soluble Tek multimer is preferably administered to a mammal that has a disease or condition mediated by angiogenesis, more preferably a solid tumor or a disease or condition characterized by ocular neovascularization.
In some embodiments the method further comprises treating the mammal with a second chemotherapeutic agent and or with radiation. The second chemotherapeutic agent may be selected from the group consisting of alkylating agents, antimetabolites, vinca alkaloids and other plant-derived chemotherapeutics, nitrosoureas, antitumor antibiotics, antitumor enzymes, topoisomerase inhibitors, platinum analogs, adrenocortical suppressants, hormones, hormone agonists, hormone antagonists, antibodies, immunotherapeutics, blood cell factors, radiotherapeutics, and biological response modifiers, and more preferably selected from the group consisting of cisplatin, cyclophosphamide, mechloretamine, melphalan, bleomycin, carboplatin, fluorouracil, 5-fluorodeoxyuridine, methotrexate, taxol, asparaginase, vincristine, and vinblastine, lymphokines and cytokines such as interleukins, interferons (including alpha, beta, or delta), and TNF, chlorambucil, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, cytarabine, mercaptopurine, thioguanine, vindesine, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, L-asparaginase, hydroxyurea, methylhydrazine, mitotane, tamoxifen, and fluoxymesterone, Flt3 ligand, CD40 ligand, interleukin-2, interleukin-12, 4-1BB ligand, anti-4-1BB antibodies, TNF antagonists and TNF receptor antagonists including TNFR/Fc, TWEAK antagonists and TWEAK-R antagonists including TWEAK-R/Fc, TRAIL, CD148 agonists, VEGF antagonists including anti-VEGF antibodies, and VEGF receptor antagonists.
The invention is further directed to a method of inhibiting the binding of a Tek ligand to Tek in a mammal in need of such treatment, comprising administering to the mammal an inhibition-effective amount of a Tek antagonist. The Tek antagonist is preferably a fragment of Tek extracellular domain, a soluble Tek multimer, or an antibody or antibody fragment.
The invention is also directed to the use of a Tek antagonist for the preparation of a medicament for inhibiting angiogenesis in a mammal in need of such treatment, or for inhibiting the binding of a Tek ligand to Tek in a mammal in need of such treatment. The Tek antagonist is preferably a fragment of Tek extracellular domain, a soluble Tek multimer, or an antibody or antibody fragment.